A survey of metronidazole and vancomycin resistance in strains of Clostridium difficile isolated in Warsaw, Poland

The drug of choice used to treat Clostridium difficile-associated diarroea (CDAD) are metronidazole and vancomycin. Information about emergence of antimicrobial resistance among C. difficile strains to metronidazole and intermediate resistance to vancomycin in some countries are alarming. This study was performed to determine the susceptibility to metronidazole and vancomycin of 193 C. difficile strains isolated in our diagnostic laboratory between year 1998 and 2003 from patients adults and children suffering from CDAD. Among these strains, 142 produced toxin A and B (TcdA(+)TcdB(+)), 43 only B (TcdA(-)TcdB(+)) and 8 were nontoxigenic. We have not observed any differences in susceptibility to metronidazole and vancomycin between all C. difficile strains under investigation (toxinogenic and non-toxinogenic). Resistance to metronidazole and vancomycin was not observed.     

Clostridium difficile and enterotoxigenic Bacteroides fragilis strains isolated from patients with antibiotic associated diarrhoea

From the fecal samples of 332 patients with a clinical diagnosis of antibiotic associated diarrhoea (AAD), 131 Clostridium difficile strains were isolated. For detection of toxin A in the isolated strains the enzymatic immunoassay was used. The cytopathic effect was determined on McCoy cell line. PCR was used for the detection of non-repeating and repeating sequences of toxin A gene and non-repeating sequences of toxin B gene. One hundred and six isolated C. difficile strains were TcdA(+)TcdB(+), 10 strains TcdA(-)TcB(+) and 15 were non-toxigenic TcdA(-)TcdB(-). Out of the same fecal samples 50 Bacteroides fragilis strains were isolated. All B. fragilis strains were tested in PCR reaction for fragilysine gene detection (bft). In 9 strains (18%) this gene was detected and the strains could be assumed as enterotoxigenic Bacteroides fragilis (ETBF). In 4 fecal samples toxigenic C. difficile (TcdA(+)TcdB(+)) was found simultaneously with ETBF. One sample contained C. difficile (TcdA(-)TcdB(+)) and ETBF. Out of 4 fecal samples only ETBF was isolated. The cytotoxicity of ETBF strains was tested on HT29/C1 human colon carcinoma cell line. The cytotoxicity titer in the range of 20 and 80 was observed.     

Molecular cloning, overexpression in Escherichia coli, and purification of 6x his-tagged C-terminal domain of Clostridium difficile toxins A and B

Genomic DNA from ribotype-01 and -17 Clostridium difficile strains was used for amplification of the sequences encoding the carboxy-terminal domain of toxins A (TcdA) and B (TcdB). The deduced C-terminal TcdB ribotype-01 and -17 domains share 99.5% amino acid sequence identity while TcdA ribotype-17 comprises a 607 amino acid deletion compared to TcdA-01. When compared to previously sequenced C. difficile toxins, 99.3% amino acid identity was found between TcdA-01 and TcdA from strain VPI10643 and 98.8% identity between TcdA-17 and TcdA from strain F-1470. The obtained sequences were fused in 3' to a sequence encoding a hexahistidine tag and cloned into an Escherichia coli expression vector. The recombinant proteins were expressed in E. coli and purified using single-step metal-chelate chromatography. The recombinant carboxy-terminal domain of TcdA-01 was purified from the soluble E. coli lysate fraction whereas TcdA-17 and TcdB-17 carboxy-terminal domains were purified from inclusion bodies. At least 40 mg of each protein was purified per liter of bacterial culture. The recombinant toxin domains were detected specifically by Western blot and ELISA with antibodies against native C. difficile toxins. This study demonstrated that the carboxy-terminal domains of TcdA and TcdB can be produced using an E. coli expression system and easily purified. These recombinant, stable, and non-toxic proteins provide a convenient source for use in the diagnosis of C. difficile infections, instead of native toxins, as controls and calibrators in immunoassay kits and to obtain specific monoclonal antibodies.     

Clostridium difficile in emergency room

Clostridium difficile strains are known as etiological agents of pseudomembranous colitis (PMC), antibiotic-associated diarrhea (AAC) and colitis (AAC) and hospital-acquired infections. The aim of this study was to determine the frequency of C. difficile infection among patients in the emergency room and to compare isolated strains by phenotypic and genotypic characteristics. During a period of 11 months, 56 stool samples taken from diarrheic patients hospitalized in the emergency room of the Medical Center UC Davis and 14 environmental samples were cultured for isolation of C. difficile strains. Eighteen C. difficile strains were isolated from stool samples cultured on selective TCCCA plates and 5 strains from environmental samples using Rodac plates. Eleven toxigenic (TcdA+/TcdB+), 6 non-toxigenic (TcdA-/TcdB-) and unique toxin A-negative/toxin B-positive (TcdA-/TcdB+) C. difficile strains were detected among patients' isolates and 3 toxigenic and 2 non-toxigenic strains-among environmental samples. The majority of C. difficile-positive patients were treated previously by antibiotics. Four strains isolated from patients' fecal samples and one strain isolated from the environment demonstrated high-level resistance to erythromycin and clindamycin (MIC >256mug/mL). The results obtained by AP-PCR and PCR-ribotyping revealed genetic heterogeneity among the strains isolated from patients' fecal samples. However, similarity was observed among environmental strains and strains isolated from patients' fecal samples. Considering the importance of emergency room patients as a potential source of C. difficile strains, it appears to be important examine these patients for C. difficile before transfer to the other hospital units.     

Comparative study of thermoresistance spores of Clostridium difficile strains belonging to different toxigenicity groups

The thermoresistance of spores of Clostridium difficile strains belonging to the different toxigenicity groups was compared in the study. Among spores of toxigenicity C. difficile strains (26 C. difficile strains produced toxins A and B (TcdA+TcdB+) and 32 C. difficile strains produced only toxin B (TcdA-TcdB+) were high thermoresistant. Between spores of non-toxigenic C. difficile strains much lower thermoresistance was observed. In conclusion, more studies are needed to clarify the importance of spores transmission in the increasing number of AAD cases in Poland.     

Clostridium difficile strains not producing Toxin A,but producing Toxin B [toxA(-)tox B(+)]--etiologic agent of antibiotic associated diarrhoea (AAD) in Poland

Previously, toxin A-negative/toxin B-positive Clostridium difficile strains were not thought to be associated with clinically significant diseases. In our study among 159 tested C. difficile strains isolated from feacal samples from 413 patients with antibiotic associated diarrhoea (AAD) 17 strains (11%) were negative in the "Culturette Brand Toxin" CD (Becton-Dickinson) for detection toxin A and positive in the TOX A/B test, designed for detection of both toxins. The conserved regions of both toxin genes were detectable in all of isolates studied by the PCR. Nine of these C. difficile strains had a deletion in the A gene and remaining 8 strains, revealed an amplicon with the expected size of approximately 2500 bp. In this paper we described the first time the toxin A-negative/toxin B-positive C. difficile strains with deletion in toxin A gene, isolated from the faecal samples of patient with AAD in Poland.     

Laboratory diagnosis of Clostridium difficile associated diarrhoea and molecular characterization of clinical isolates

We evaluated a three-step algorithm for laboratory diagnosis of Clostridium difficile-associated diarrhoea (CDAD). First, stool specimens were screened using an EIA test for glutamate dehydrogenase detection. Screen-positive specimens were tested by a rapid cytotoxintoxin A/B assay and subjected to stool culture. All cultures positive for C. difficile underwent toxigenic culture. The results showed that toxigenic culture allowed us to recover 37/156 (24.4%) stool samples harbouring toxigenic C. difficile that would have been missed by using faecal cytotoxin assay alone. This determined an increase in infection prevalence of 4.2% (from 11.4% to 15.6 %). Furthermore, to characterize the clinical Clostridium difficile isolates and the distribution of PCR ribotypes circulating in the San Carlo Borromeo hospital, molecular typing using semi-automated repetitive-sequence-based PCR (rep- PCR) and PCR ribotyping, and an evaluation of the antibiotic resistance were also performed. Among them, 71 indistinguishable strains were detected by rep-PCR and 83 by PCR-ribotyping revealing C. difficile outbreaks in our hospital. A total of 6 different ribotypes were obtained by PCR ribotyping. The most frequent ribotype was 018 (88.2%) that also showed resistance to moxifloxacin. In one case, uncommon PCR ribotype 186 was also identified.     

Clostridium difficile-associated diarrhea from a general hospital in Argentina

Clostridium difficile is responsible for 15-25% of all cases of antibiotic associated diarrhea. The incidence of infection with this organism is increasing in hospitals worldwide, consequent to the widespread use of broad-spectrum antibiotics. Although the clinical and financial impact of nosocomial C. difficile infection is believed to be significant, only limited information is available on the importance of C. difficile as a cause of diarrhea in Argentina. The aim of the study was to evaluate the impact and diagnosis methods of CDAD from symptomatic patients in a general hospital from Argentina. Consecutive diarrheal stool samples from symptomatic patients from a General Hospital in Argentina were screened for toxigenic C. difficile between April 2000 and April 2001. Toxins were detected in stools by the Premier Cytoclone A+B EIA. Each specimen was examined for toxigenic C. difficile strains by culture. From 104 specimens, 40 (38.5%) [32 of 87 patients (36.8%)] were positive and 64 (61.5%) [55 of 87 patients (63.2%)] were negative by stool toxin assay and/or toxigenic culture. In 11 of 40 positives samples C. difficile toxins were detected only by toxigenic culture. Five (15.6%) patients presented with symptomatic recurrences. Toxin-negative strains were not isolated. This data indicates that the high prevalence of toxigenic strains of C. difficile is of concern in routine diagnostic testing for C. difficile toxins in our study population. Detection of toxins in stools by EIA, coupled with testing strains for toxigenicity only in those cases in which direct toxin assay produces negative results, may be a satisfactory strategy. CDAD is an emerging nosocomial problem in our hospital. It will be necessary to evaluate the epidemiology and measures to control nosocomial spread.     

Binary toxin producing Clostridium difficile strains

Clostridium difficile produces three toxins, TcdA, TcdB and CDT. TcdA and TcdB are single-stranded molecules acting as glucosyltransferases specific for small GTPases. CDT is an actin specific ADP-ribosylating binary toxin characteristically composed of two independent components, enzymatic CDTa (48 kDa) and binding CDTb (99 kDa). The cdtA and cdtB genes were sequenced in two CDT-positive strains of C. difficile (CD 196 and 8864) and at least two CDT-negative strains with truncated form of binary toxin genes are known (VPI 10463 and C. difficile genome strain 630). The prevalence of binary toxin producing strains is estimated to be from 1.6% to 5.5%, although a much higher proportion has been reported in some studies. The role of the binary toxin as an additional virulence factor is discussed.     

The repetitive oligopeptide sequences modulate cytopathic potency but are not crucial for cellular uptake of Clostridium difficile toxin A

The pathogenicity of Clostridium difficile is primarily linked to secretion of the intracellular acting toxins A (TcdA) and B (TcdB) which monoglucosylate and thereby inactivate Rho GTPases of host cells. Although the molecular mode of action of TcdA and TcdB is well understood, far less is known about toxin binding and uptake. It is acknowledged that the C-terminally combined repetitive oligopeptides (CROPs) of the toxins function as receptor binding domain. The current study evaluates the role of the CROP domain with respect to functionality of TcdA and TcdB. Therefore, we generated truncated TcdA devoid of the CROPs (TcdA(1-1874)) and found that this mutant was still cytopathic. However, TcdA(1-1874) possesses about 5 to 10-fold less potency towards 3T3 and HT29 cells compared to the full length toxin. Interestingly, CHO-C6 cells even showed almost identical susceptibility towards truncated and full length TcdA concerning Rac1 glucosylation or cell rounding, respectively. FACS and Western blot analyses elucidated these differences and revealed a correlation between CROP-binding to the cell surface and toxin potency. These findings refute the accepted opinion of solely CROP-mediated toxin internalization. Competition experiments demonstrated that presence neither of TcdA CROPs nor of full length TcdA reduced binding of truncated TcdA(1-1874) to HT29 cells. We assume that toxin uptake might additionally occur through alternative receptor structures and/or other associated endocytotic pathways. The second assumption was substantiated by TER measurements showing that basolaterally applied TcdA(1-1874) exhibits considerably higher cytotoxic potency than apically applied mutant or even full length TcdA, the latter being almost independent of the side of application. Thus, different routes for cellular uptake might enable the toxins to enter a broader repertoire of cell types leading to the observed multifarious pathogenesis of C. difficile.     

Surface proteins from Lactobacillus kefir antagonize in vitro cytotoxic effect of Clostridium difficile toxins

In this work, the ability of S-layer proteins from kefir-isolated Lactobacillus kefir strains to antagonize the cytophatic effects of toxins from Clostridium difficile (TcdA and TcdB) on eukaryotic cells in vitro was tested by cell detachment assay. S-layer proteins from eight different L. kefir strains were able to inhibit the damage induced by C. difficile spent culture supernatant to Vero cells. Besides, same protective effect was observed by F-actin network staining. S-layer proteins from aggregating L. kefir strains (CIDCA 83115, 8321, 8345 and 8348) showed a higher inhibitory ability than those belonging to non-aggregating ones (CIDCA 83111, 83113, JCM 5818 and ATCC 8007), suggesting that differences in the structure could be related to the ability to antagonize the effect of clostridial toxins. Similar results were obtained using purified TcdA and TcdB. Protective effect was not affected by proteases inhibitors or heat treatment, thus indicating that proteolytic activity is not involved. Only preincubation with specific anti-S-layer antibodies significantly reduced the inhibitory effect of S-layer proteins, suggesting that this could be attributed to a direct interaction between clostridial toxins and L. kefir S-layer protein. Interestingly, the interaction of toxins with S-layer carrying bacteria was observed by dot blot and fluorescence microscopy with specific anti-TcdA or anti-TcdB antibodies, although L. kefir cells did not show protective effects. We hypothesize that the interaction between clostridial toxins and soluble S-layer molecules is different from the interaction with S-layer on the surface of the bacteria thus leading a different ability to antagonize cytotoxic effect. This is the first report showing the ability of S-layer proteins from kefir lactobacilli to antagonize biological effects of bacterial toxins. These results encourage further research on the role of bacterial surface molecules to the probiotic properties of L. kefir and could contribute to strain selection with potential therapeutic or prophylactic benefits towards CDAD.     

Protection from Clostridium difficile toxin B-catalysed Rac1/Cdc42 glucosylation by tauroursodeoxycholic acid-induced Rac1/Cdc42 phosphorylation

Toxin A (TcdA) and toxin B (TcdB) are the major virulence factors of Clostridium difficile-associated diarrhoea (CDAD). TcdA and TcdB mono-glucosylate small GTPases of the Rho family, thereby causing actin re-organisation in colonocytes, resulting in the loss of colonic barrier function. The hydrophilic bile acid tauroursodeoxycholic acid (TUDCA) is an approved drug for the treatment of cholestasis and biliary cirrhosis. In this study, TUDCA-induced activation of Akt1 is presented to increase cellular levels of pS71-Rac1/Cdc42 in human hepatocarcinoma (HepG2) cells, showing for the first time that bile acid signalling affects the activity of Rho proteins. Rac1/Cdc42 phosphorylation, in turn, protects Rac1/Cdc42 from TcdB-catalysed glucosylation and reduces the TcdB-induced cytopathic effects in HepG2 cells. The results of this study indicate that TUDCA may prove useful as a therapeutic agent for the treatment of CDAD.     

Clostridium difficile toxins: more than mere inhibitors of Rho proteins

Toxin A (TcdA) and Toxin B (TcdB) are the major pathogenicity factors of the Clostridium difficile-associated diarrhoea (CDAD). The single-chained protein toxins enter their target cells by receptor-mediated endocytosis. New data show the critical role of auto-catalytic processing for target cell entry. Inside the cell, the toxins mono-glucosylate and thereby inactivate low molecular mass GTP-binding proteins of the Rho subfamily. Toxin-treated cells respond to RhoA glucosylation with up-regulation and activation of the pro-apoptotic Rho family protein RhoB. These data reinforce the critical role of the glucosyltransferase activity for programmed cell death and show that TcdA and TcdB, generally classified as broad-spectrum inhibitors of Rho proteins, are also capable of activating Rho proteins.     

Porcine and bovine <Emphasis Type="Italic">Clostridium difficile</Emphasis> ribotype 078 isolates demonstrate similar growth and toxigenic properties

Clostridioides (C.) difficile are found in cows, pigs and poultry suggesting that this pathogen is present and more importantly animals could act as a reservoir, via food or environment, of human C. difficile infection. Molecular methods together with phenotypical characterisation bring integrated and important tools to describe diversity and nature of bacteria including C. difficile. Moreover, similar or identical C. difficile types are found in different farm animals. This study aimed to phenotypically characterise C. difficile isolates belonging to ribotype 078 and to identify differences such as growth and toxicity between porcine and bovine isolates. C. difficile isolates were assessed for the growth behaviour (turbidimetry), metabolic potential (Biolog AN) and toxin production (ELISA method) in vitro. The concentration of released either toxin A (TcdA) or toxin B (TcdB) varied greatly between the isolates tested; however, it did not differ between the porcine and bovine ribotypes. Also, the TcdA/TcdB ratio of the isolates did not show a difference either. The most common metabolised substrates were pyruvic acid followed by α-ketobutyric acid. The results show that both porcine and bovine C. difficile RT 078 share similar phenotypical characteristics including growth and production of toxins. The findings may help understand the virulence of C. difficile RT 078 in porcine and bovine species.     

Conformational Analysis of Clostridium difficile Toxin B and Its Implications for Substrate Recognition

Clostridium difficile (C. difficile) is an opportunistic pathogen that can cause potentially lethal hospital-acquired infections. The cellular damage that it causes is the result of two large clostridial cytotoxins: TcdA and TcdB which act by glucosylating cytosolic G-proteins, mis-regulation of which induces apoptosis. TcdB is a large flexible protein that appears to undergo significant structural rearrangement upon accommodation of its substrates: UDP-glucose and a Rho-family GTPase. To characterize the conformational space of TcdB, we applied normal mode and hinge-region analysis, followed by long-timescale unbiased molecular dynamics. In order to examine the TcdB and RhoA interaction, macromolecular docking and simulation of the TcdB/RhoA complex was performed. Generalized Masked Delaunay analysis of the simulations determined the extent of significant motions. This combination of methods elucidated a wide range of motions within TcdB that are reiterated in both the low-cost normal mode analysis and the extensive MD simulation. Of particular interest are the coupled motions between a peripheral 4-helix bundle and a small loop in the active site that must rearrange to allow RhoA entry to the catalytic site. These extensive coupled motions are indicative of TcdB using a conformational capture mechanism for substrate accommodation.     

High temporal resolution of glucosyltransferase dependent and independent effects of Clostridium difficile toxins across multiple cell types

Background

Clostridium difficile toxins A and B (TcdA and TcdB), considered to be essential for C. difficile infection, affect the morphology of several cell types with different potencies and timing. However, morphological changes over various time scales are poorly characterized. The toxins’ glucosyltransferase domains are critical to their deleterious effects, and cell responses to glucosyltransferase-independent activities are incompletely understood. By tracking morphological changes of multiple cell types to C. difficile toxins with high temporal resolution, cellular responses to TcdA, TcdB, and a glucosyltransferase-deficient TcdB (gdTcdB) are elucidated.

Results

Human umbilical vein endothelial cells, J774 macrophage-like cells, and four epithelial cell lines (HCT8, T84, CHO, and immortalized mouse cecal epithelial cells) were treated with TcdA, TcdB, gdTcdB. Impedance across cell cultures was measured to track changes in cell morphology. Metrics from impedance data, developed to quantify rapid and long-lasting responses, produced standard curves with wide dynamic ranges that defined cell line sensitivities. Except for T84 cells, all cell lines were most sensitive to TcdB. J774 macrophages stretched and increased in size in response to TcdA and TcdB but not gdTcdB. High concentrations of TcdB and gdTcdB (>10 ng/ml) greatly reduced macrophage viability. In HCT8 cells, gdTcdB did not induce a rapid cytopathic effect, yet it delayed TcdA and TcdB’s rapid effects. gdTcdB did not clearly delay TcdA or TcdB’s toxin-induced effects on macrophages.

Conclusions

Epithelial and endothelial cells have similar responses to toxins yet differ in timing and degree. Relative potencies of TcdA and TcdB in mouse epithelial cells in vitro do not correlate with potencies in vivo. TcdB requires glucosyltransferase activity to cause macrophages to spread, but cell death from high TcdB concentrations is glucosyltransferase-independent. Competition experiments with gdTcdB in epithelial cells confirm common TcdA and TcdB mechanisms, yet different responses of macrophages to TcdA and TcdB suggest different, additional mechanisms or targets in these cells. This first-time, precise quantification of the response of multiple cell lines to TcdA and TcdB provides a comparative framework for delineating the roles of different cell types and toxin-host interactions.

Electronic supplementary material

The online version of this article (doi:10.1186/s12866-015-0361-4) contains supplementary material, which is available to authorized users.     

Structural determinants of Clostridium difficile toxin A glucosyltransferase activity

The principle virulence factors in Clostridium difficile pathogenesis are TcdA and TcdB, homologous glucosyltransferases capable of inactivating small GTPases within the host cell. We present crystal structures of the TcdA glucosyltransferase domain in the presence and absence of the co-substrate UDP-glucose. Although the enzymatic core is similar to that of TcdB, the proposed GTPase-binding surface differs significantly. We show that TcdA is comparable with TcdB in its modification of Rho family substrates and that, unlike TcdB, TcdA is also capable of modifying Rap family GTPases both in vitro and in cells. The glucosyltransferase activities of both toxins are reduced in the context of the holotoxin but can be restored with autoproteolytic activation and glucosyltransferase domain release. These studies highlight the importance of cellular activation in determining the array of substrates available to the toxins once delivered into the cell.     

Identification and susceptibility to antimicrobial agents of strictly anaerobic bacteria isolated from hospitalized patients

The aim of this study was to identify anaerobic strains isolated in 2001 from clinical specimens obtained from patients of Warsaw hospital and to evaluate a susceptibility of these strains to antimicrobial agents. In 2001 two hundred and twenty five clinical strains of obligate anaerobes were cultured, which were identified in the automatic ATB system (bioMérieux, France) using biochemical tests API 20 A. Drug-susceptibility of strains was determined also in ATB system with the use of ATB ANA strips. C. difficile strains were isolated on selective CCCA medium. Toxins A/B of C. difficile directly in stool specimens were detected by means of ELISA test (TechLab, USA). Fifty four strains of Gram-negative anaerobes (B. fragilis strains dominated) and 171 strains of Gram-positive anaerobes (the greatest number of strains belonged to genus Peptostreptococcus) were cultured from clinical specimens. In the cases of antibiotic-associated diarrhea 28 C. difficile strains were isolated and C. difficile toxins A/B were detected in 39 stool samples. The most active in vitro antimicrobials against Gram-negative anaerobes were metronidazole, imipenem, ticarcillin combined with clavulanic acid and piperacillin with tazobactam. Gram-positive, clinical strains of anaerobes were the most susceptible in vitro to beta-lactam antibiotics combined with beta-lactamase inhibitors (amoxicillin/clavulanate, piperacillin/tazobactam, ticarcillin/clavulanate) and imipenem.     

Heterogeneity of large clostridial toxins: importance of Clostridium difficile toxinotypes

Clostridium difficile toxinotypes are groups of strains defined by changes in the PaLoc region encoding two main virulence factors: toxins TcdA and TcdB. Currently, 24 variant toxinotypes (I-XXIV) are known, in addition to toxinotype 0 strains, which contain a PaLoc identical to the reference strain VPI 10463. Variant toxinotypes can also differ from toxinotype 0 strains in their toxin production pattern. The most-studied variant strains are TcdA-, TcdB+ (A-B+) strains and binary toxin CDT-producing strains. Variations in toxin genes are also conserved on the protein level and variant toxins can differ in size, antibody reactivity, pattern of intracellular targets (small GTPases) and consequently in their effects on the cell. Toxinotypes do not correlate with particular forms of disease or patient populations, but some toxinotypes (IIIb and VIII) are currently associated with disease of increased severity and outbreaks worldwide. Variant toxinotypes are very common in animal hosts and can represent from 40% to 100% of all isolates. Among human isolates, variant toxinotypes usually represent up to 10% of strains but their prevalence is increasing.     

The structure of Clostridium difficile toxin A glucosyltransferase domain bound to Mn(2+) and UDP provides insights into glucosyltransferase activity and product release

Clostridium?difficile toxin?A (TcdA) is a member of the large clostridial toxin family, and is responsible, together with C.?difficile toxin?B (TcdB), for many clinical symptoms during human infections. Like other large clostridial toxins, TcdA catalyzes the glucosylation of GTPases, and is able to inactivate small GTPases within the host cell. Here, we report the crystal structures of the TcdA glucosyltransferase domain (TcdA-GT) in the apo form and in the presence of Mn(2+) and hydrolyzed UDP-glucose. These structures, together with the recently reported crystal structure of TcdA-GT bound to UDP-glucose, provide a detailed understanding of the conformational changes of TcdA that occur during the catalytic cycle. Indeed, we present a new intermediate conformation of a so-called 'lid' loop (residues?510-522 in TcdA), concomitant with the absence of glucose in the catalytic domain. The recombinant TcdA was expressed in Brevibacillus in the inactive apo form. High thermal stability of wild-type TcdA was observed only after the addition of both Mn(2+) and UDP-glucose. The glucosylhydrolase activity, which is readily restored after reconstitution with both these cofactors, was similar to that reported for TcdB. Interestingly, we found that ammonium, like K(+) , is able to activate the UDP-glucose hydrolase activities of TcdA. Consequently, the presence of ammonium in the crystallization buffer enabled us to obtain the first crystal structure of TcdA-GT bound to the hydrolysis product UDP. Database ??Coordinates of apo-TcdA-GT and Mn(2+) -UDP-TcdA-GT are available in the Protein Data Bank under the accession numbers 4DMV and 4DMW, respectively.